Hepatocytes and renal tubular epithelial cells tend to shed flavivirus long after the acute phase has been resolved. In these cells, flavivirus replicates in autophagosomes, which are upregulated by the virus. The mechanism is unknown. We find that Dengue-2 or Modoc (a murine flavivirus) NS4A is the sole viral protein upregulating autophagy, and that autophagy is important to virus replication in epithelial cells. NS4A-induced autophagy protects infected cells against death, a prerequisite for the establishment of viral persistence. We propose to explore how NS4A induces autophagy, increase in viral production, and establishment of persistent infection. We hypothesize that NS4A insertion into the ER induces ER stress, leading to induction of ROS. This increased ROS then activates the ATM pathways, thus downregulating M-TOR and leading to autophagy. Our hypothesis of a linear pathway is certainly oversimplified but starting with a simple model and expanding to incorporate complications will be more productive than attempting a potentially overwhelming analysis. For these investigations we propose to: AIM I: CHARACTERIZE CELLULAR AND VIRAL COMPONENTS LEADING TO AUTOPHAGY AFTER VIRAL INFECTION. We will first ask if what we see with Dengue 2 and NS4A is unique to this strain of Dengue or if NS4A functions similarly in all forms of Dengue. A: Determine if the autophagy and subsequent protection of cells from death produced by Dengue-2 is generalizable to all Dengue strains? B: Identify the domain(s) of Dengue NS4A responsible for inducing autophagy. By finding which part of this molecule is responsible for this induction we can then attempt to block it, thus interfering with the ability of the virus to survive and persist. AIM II: INVESTIGATE THE MECHANISMS BY WHICH DENGUE INDUCES AUTOPHAGY. Here we will test each step of our hypothesis. We will: A: Confirm the involvement of ATM and M-TOR pathways in NS4A-induced autophagy. B: Establish if NS4A expression leads to ER stress. C: Determine whether NS4A infection leads to ROS production. D: Identify the domain of NS4A responsible for ER stress. E: Use yeast as a complementary model to determine the regulatory pathway involved in NS4A-mediated autophagy. Our findings should lead to an understanding of how flavivirus persists in its host, thus suggesting a target and potential means of reducing viral load and persistence. Furthermore, since we directly examine a means by which autophagy is regulated, these studies should lead to new insights into the means of activating and suppressing autophagy, an important question in cell biology and a potential therapeutic target in many other diseases in which autophagy is critical for cell survival, such as in neurodegenerative diseases and cancer. PUBLIC HEALTH RELEVANCE: Dengue virus, currently the world's most common mosquito-borne illness, is the leading cause of children's hospitalization in Southeast Asia and is the cause of significant morbidity and mortality. We find that flavivirus induces autophagy, preventing suicide of the host cell, leading to increased production and persistence of virus;these activities are produced by the viral protein NS4A. We wish to analyze the mechanism by which NS4A works. Understanding the mechanism should provide a means of controlling the virus and give us another option to protect nerve cells or destroy cancer cells.